Converting the specificity of trypsin to that of chymotrypsin has been
shown to require the exchange of amino acids in multiple portions of
the protein, including two surface loops which do not directly contact
the substrate. Crystallographic analysis of two mutant trypsins posse
ssing chymotrypsin-like specificity now reveals that these distal surf
ace loops alter function by directly determining the structure of the
primary binding site. Efficient acylation of cognate substrates correl
ates with a distinct backbone conformation of the conserved Gly216 res
idue. This amino acid is located on the surface of the specificity poc
ket and forms two main-chain hydrogen bonds with a nonspecific portion
of substrate. By contrast, the improvement in substrate binding affin
ity effect by the substitution of the distal Tyr172 residue with Trp d
erives from structural rearrangements at the extreme base of the pocke
t. Together, the kinetic and crystallographic data strongly suggest th
at both Asp189 and Gly216 must be considered as primary determinants o
f substrate specificity in trypsin.